ABSTRACT
The mean osmolar concentration (expressed as mM. NaCl/l.) of the serum for Lampetra planeri was found to be no mM. in the ammocoete and 113 mM. in the adult. For adult L. fiuviatilis szerum in November the mean osmolar concentration was 143 mM. and in March 136 mM.
In adult planeri the Cl concentration of the body fluids accounted for nearly 90% of the total concentration as compared with only 58% in the serum of the ammocoete. Adult fiuviatilis kept in tap water throughout the winter appeared to show an increase in Cl levels.
Evidence has been produced to support the view that the differences observed in water, fat and Cl content of ammocoete and adult planeri and adult fiuviatilis are consequences of the period of starvation preceding sexual maturity.
Pronounced seasonal variations have been found in the fat and Cl content of ammocoetes of planeri.
The osmotic uptake of water has been determined for adults of both species in fresh water. In planeri the rate of uptake for unit body weight is about four times as great as in fiuviatilis.
Observations have also been made on the osmotic loss of water of ammocoete and adult planeri and of adult fiuviatilis in sea water of different concentrations. For unit body weight osmotic loss of water in planeri is about twice that of fiuviatilis.
Total losses of Cl have been determined for ammocoete and adult planeri in distilled water and their effects on internal Cl levels have been studied. Observations have also been made on the active uptake of Cl.
Acclimatization experiments on ammocoetes and adult planeri and on adult fiuviatilis have failed to produce any evidence of regulation in sea water.
I. INTRODUCTION AND METHODS
Although we have some information on osmotic conditions in the migratory lampreys Lampetra fluviatilis and Petromyzon marinus, no observations on the purely fresh-water form L. planeri appear to have been recorded. The main purpose of the work reported in this paper was to study the problem of osmotic regulation in fresh water and most of the observations have been made on adults and ammocoetes of L. planeri taken from streams in Somerset. In some instances, however, it has been possible to make comparisons with adults of L.fluviatils fished from the River Dee in October and kept in tap water in the laboratory until the following spring.
Osmolar concentrations were determined by the vapour-pressure method, using a Baldes thermocouple and Zemicke moving-coil galvanometer, and the results have been expressed throughout in terms of millimolar NaCl solution. To facilitate comparison, freezing-point data from other workers have been recalculated in the same terms.
For the Cl analyses of body fluids and tissues extracts volumes of the order of 0·005 ml. were taken up in a fine capillary mounted against a scale. These were then delivered into small silica crucibles and standard AgNO3 solution, made up in 80% HNO3, was added to them in known volume from an Agla syringe pipette. After standing in the dark for some hours, ether and a few crystals of finely powdered ferric alum indicator were added and titration was then carried out in the same vessel, using N/50 or N/100 KCNS solution delivered from the syringe pipette. The concentration of Cl was then determined by drawing up to the same mark on the capillary a standard solution of NaCl of similar concentration to the unknown and repeating the titration as for the test sample. Using these methods Cl concentration may be determined within 1–2%.
Water content was determined on whole animals, dried in an oven at 105° C. for 24 hr. The finely powdered dried material was then used for fat estimation by extracting it with several changes of ether, followed by petroleum ether. For the determination of total Cl on the dried tissues it was found satisfactory to digest the material with N/3O-H2SO4 in sealed 5 ml. ampoules placed in an oven at 100° C. for 24 hr. 2 ml. of the dilute acid were added to each sample of about too mg., and the subsequent titrations with AgNO3 and KCNS were carried out on aliquot portions of this fluid. Because of the variations in the fat content of the dried material it was found necessary to express both water and Cl content in terms of the fat-free tissues.
Determinations of osmolar and Cl concentrations were usually made on serum, but in adult planeri it was often difficult to obtain sufficient blood for both determinations, and for this reason the peritoneal fluid was sometimes employed. Double determinations on the serum and peritoneal fluid of six animals showed good agreement in total concentration, but there was a tendency for the Cl concentration to be slightly higher in the peritoneal fluid. Where muscle extracts were used, these were prepared by grinding up the tissue in a mortar with quartz sand followed by centrifuging to obtain a clear solution. Determinations were carried out within 20 min. of preparation.
In the experiments involving weight changes the animals were folded gently into a soft absorbent cloth and afterwards weighed in water. Tests on animals which had been wiped off more roughly showed no evidence of increased permeability to water, neither did they appear to lose Cl at a greater rate than animals not subjected to such treatment. For these reasons it is not thought likely that the procedure normally employed would cause any serious skin damage. The accuracy of these weighings may be estimated as about o·2% for planeri and rather less for fluviatilis.
In the work on the uptake and loss of chloride, water analyses were carried out by the methods described by Krogh (1937 a), involving the concentration of a 50 ml. sample to about 0·5 ml. and the subsequent titration of the residue by a modified Volhard method. Although the uptake of the Cl ion may well be secondary to the uptake of Na (Ussing, 1949), Cl determinations alone have been made throughout to simplify the analytical procedures.
Where animals have been depleted of Cl by washing out in distilled water, the volumes used have always been large in relation to the size of the animal (not less than 30 ml./g.), and this water was changed every day. The distilled water was obtained fresh from a large hard glass still and, at least with the analytical methods employed here, contained no detectable quantities of Cl.
II. OSMOTIC CONDITIONS IN NORMAL ANIMALS
(a) Water and fat content
It became apparent at the outset that, while there were considerable differences in water content between adults of planeri and fluviatilis, between the ammocoete and adult of planeri, and even between ammocoetes caught at different times of the year, these differences were, to a great extent, attributable to variations in the fat content of the dried material. When this was discounted by calculating the water content as a percentage of the fat-free tissues both the range of variation within the various groups and the differences in the mean values were reduced (Table 1). Nevertheless, for planeri, fat-free values in the adult appear to be significantly higher than in the ammocoete, and in fluviatilis there are indications of an increase in the water content of the body during the period between November and March.
Ammocoetes kept for long periods in tap water have given higher values than freshly caught animals, and there seems little doubt that the raised water content of the sexually mature adult is associated with the prolonged starvation which precedes spawning. Fat content, especially in planeri, is very variable, and in the ammocoete there are marked local and seasonal variations. Fig. 1 records the observations made on ammocoetes collected at intervals throughout the year within a stretch of about half a mile in a small stream. In spite of the very great scatter of the individual observations the general trend is clear—a rapid rise in fat content during the spring from a minimum mean value of 5·5 % in mid-March to the maximum value of 36% in the middle of May. This period also coincides with the period of maximum growth in length of the ammocoete (Hardisty, unpublished) and is no doubt associated with a spring peak in the algal plankton. During the summer there appears to be little further fat accumulation, and, indeed, in August and September values were somewhat lower than those recorded in May, June and July.
For adult planeri very low fat values have been recorded towards the end of the spawning season and particularly in spent animals. On the other hand, values for animals caught in late March and early April before spawning were similar to those observed in adults of fluviatilis in March.
(b) Osmolar concentration and Cl concentration of body fluids and muscle extracts
Mean values for the osmolar concentration of the serum are similar in both ammocoete and adult planeri, although in both the observations cover a wide range of individual values (Table 2). The higher values found for the serum of fluviatilis are similar to those recorded by Galloway (1933) and Deckhuyzen (1904) for this species. Determinations on six animals in March gave slightly lower values than those obtained in November.
The mean chloride concentration of serum and peritoneal fluid from adult planeri is nearly twice that of the ammocoete and accounts for no less than 89% of the total concentration. For fluviatilis in November the mean Cl concentration represents only 79% of the osmolar concentration, but in the animals killed in March this ratio was increased to a value much closer to that of the adult planeri (85%). These Cl values, although in good agreement with those of Galloway (1933), are considerably greater than Robertson’s (1954) values for the serum Cl of this animal in February. However, it is noteworthy that the total ionic concentration of Robertson’s animals suggested a lower level of osmolar concentration than that observed here. In Petromyzon marinus, which ascends the rivers in the spring, the osmolar concentration appears to be distinctly higher than influviatilis, although the Cl fraction forms a similar percentage of the total concentration.
Determinations of the total Cl content of the dried tissues of whole animals after fat extraction indicate that, in planeri, the mean Cl content of the adult is more than twice that of the ammocoete taken from the stream in spring and early summer. Assuming all the Cl to be osmotically active and expressing the results as mM./kg. of body water, the overall concentration of this ion in the adult works out at about twice that of the ammocoete, a result which agrees quite well with the direct determinations of the Cl concentration of serum and muscle extracts. Values obtained for fluviatilis in November were lower than in the adult of planeri, but considerably higher figures were recorded for animals killed in March. These observations, taken in conjunction with the information on Cl concentration, suggest that in both species starvation is accompanied by an increase in the Cl content of the animals. This has been confirmed for the ammocoete by keeping animals, caught in September, for a period of 3 weeks in tap water in the laboratory and comparing their Cl content with that of control animals which had been taken from the stream at the same time and killed immediately (Table 3). In spite of the slightly raised water content which has been noted as a normal accompaniment of starvation, these Cl figures for the fasting ammocoetes indicate an overall Cl concentration of 37 mM./kg. body water as compared with 33 mM. for the control group. A marked increase in blood Cl has, in fact, been observed in ammocoetes kept for much longer periods in tap water. Thus three large animals after 2 months starvation gave values of 78, 84 and 86 mM. Cl, and a further specimen kept in the laboratory for 6 months gave 88 mM. ; figures which approach the lower range of adult values.
Seasonal changes in the total Cl content of ammocoetes have been followed in those animals which were collected throughout the year for fat determinations. All the animals used were between 70 and 100 mm. long, and, although they were collected from a restricted stretch of the stream, the range of variation in Cl content is considerable. Values plotted in Fig. 1 are for the pooled material from two to four animals. The extent of the seasonal changes in Cl levels was as much as 60%, with maximum mean values of 200 and 210 mM. Cl/kg. dry material in March and September and minimum values of 130–140 mM. for animals collected in April, June and early July. Taking into account the changes in water content which also occur, the corresponding overall Cl concentrations would fall from a maximum value of 37 mM. Cl/kg. body water in mid-March to a minimum of 24mM. in June. It would be interesting to know whether significant seasonal variations occur also in the Cl concentration of the ammocoete serum. Although examination of the individual observations failed to reveal any close negative correlation between fat content and Cl levels, the general trend of the seasonal changes is suggestive of some relationship between nutritional state and Cl content. Minimum Cl values were observed in early summer when the fat content of the animal had reached its peak and the period of most rapid fat accumulation in early spring coincides with a drop in Cl levels. During the late summer and autumn the spread of the fat values appears to increase with perhaps a slight downward trend, and throughout this period Cl figures show a marked increase. The drop in Cl content in the spring may reflect a displacement of inorganic by organic metabolites in the body fluids, as a consequence of the abundant supply of food available at this time, but it is more difficult to understand why Cl levels should rise steeply later in the summer when feeding is presumably still quite active.
In both ammocoete and adult muscle extracts the Cl concentration is approximately half that observed in the serum, and the values observed agree quite well with those calculated from the Cl determinations made on the dried tissues. Although determinations of the osmolar concentration of muscle extracts have been carried out, little importance is attached to the absolute values in view of the observations of McCormack (1953) on the rapid increase in total concentration of tissue extracts maintained at o°C. It may, however, be worth recording that the values obtained for ammocoete muscle extracts (mean 144 mM.) were much lower than for the muscle of the adult planeri or fluviatilis (mean 199 mM.), and that these differences are not accounted for entirely by differences in the Cl concentration of these extracts.
III. THE CHLORIDE BALANCE
(a) Chloride losses and their effects on the concentration of the body fluids
Observations on the rate of loss of Cl ions from adult planeri kept in distilled water showed great individual variations. For the fifteen animals tested, total losses over periods of 4–8 days varied from 9 to 85 μM. Cl, representing daily rates of 1–20μM. For nine of the experimental animals, however, the daily rate was below 3μM. and for seven animals which were previously weighed the mean daily rate of loss/g. of body wt. was 1·8μM. Taking the mean Cl content of the fresh adult tissues as 49μM./g., this implies a daily loss of nearly 4% of the total Cl in the body, and yet in spite of this rate of loss adults have survived in distilled water for 14 days and ammocoetes up to 21 days. Tests carried out on twelve ammocoetes gave rates varying from 0·2 to 1·2μM./g./day, and it may well be that a somewhat lower rate of Cl depletion is responsible for the greater tolerance shown by the ammocoete to distilled water. The highest rates of loss of Cl were generally observed during the first day, reaching minimum values by the third or fourth day and remaining at a low level for the rest of the period.
The effects of Cl depletion on the concentration of the peritoneal fluid were investigated by making Cl determinations on six adult planeri before and after washing out in distilled water. At the same time actual Cl losses were measured by Cl analyses of the distilled water (Table 4). Knowing the weight of these animals it was possible to estimate the overall effect of the Cl losses, assuming that the Cl ion is taken equally from body fluids and tissues. Except in one instance, however, the observed decrease in concentration of the peritoneal fluid was considerably greater than the calculated value, implying a relatively greater rate of loss of Cl from the body fluids as compared with the tissues. In spite of the serious reductions in concentration which were observed in these animals, all except one survived the period of washing out. The exception was the animal which had shown an abnormally high rate of loss (51μM.) and a 50% reduction in Cl concentration. This animal died during the fourth day in distilled water.
Cl estimations have also been carried out on ammocoetes after 14 days washing out in distilled water (Table 5), and the results show a fall in concentration of the same order as that calculated from the known rates of loss of Cl for ammocoetes. The mean percentage reduction in Cl concentration (10%) was less than that observed in the adults which had been washed out in distilled water for 7 days.
Since Cl accounts for nearly 90% of the osmolar concentration of the adult body fluids, the drop in Cl concentration observed in distilled water must inevitably produce a comparable reduction in the total concentration, and indeed decreases of 18 and 29% have been observed in the osmolar concentration of the peritoneal fluid of two adults which had been washed out for 7 days. However, serious as these losses are in their effects on internal concentration, it does not appear that in regard to Cl retentivity planeri compares unfavourably with those freshwater teleosts for which similar information is available. Thus for Ameirus and Gasterosteos aculeatus Krogh (1937a) gives figures which indicate rates of loss of about 5 μM./g./day, while even the eel (Callamand, 1943; Krogh, 1939), whose ability to resist starvation in fresh water seems to depend entirely on Cl retention, may lose from 0·2 to 1·8μM./g./day. For amphibia, Krogh (1937b) found rates of 0·7–3·0μM./g./day in Rana esculenta, and for Amblystoma kept in millimolar NaCl solutions Jorgensen, Levi & Ussing (1947) reported mean rates of 4μM./g./day. In making such comparisons, however, it must be borne in mind, particularly where animals are known to have a well-developed capacity for active uptake of Cl ions, that such figures may only represent the net rate of Cl loss, i.e. the resultant of loss and uptake of Cl occurring simultaneously. Nevertheless, with the volumes of distilled water employed here it does not seem likely that active uptake could very seriously reduce the concentration of the water, and it is thought unlikely that the absolute rate of Cl loss is of a higher order altogether than the net rates observed. Wikgren (1953) estimated the absolute rate of loss of fluviatilis as about 12–24 μM./g./day, of which only about 1 % is lost in the urine. In these experiments, which were generally of short duration, the animals were kept in a smaller volume of distilled water (less than 10 ml./g. of animal), and loss and uptake of Cl were followed by making frequent analyses on the same water. Taking the mean Cl content of fluviatilis from Table 2, such losses would imply a depletion of about 70% of the total Cl in the body in 24 hr. It is hard to believe that a similar rate of Cl depletion can obtain in planeri in view of the tolerance shown to distilled water over long periods. In the course of the experiment summarized in Table 5, eighteen ammocoetes with a total weight of about 50 g. were kept in a vessel containing 5 l. of freshly distilled water, which was kept running at a rate of approximately 200 ml./hr. Even if uptake could take place under such circumstances it is hardly conceivable that it would substantially reduce the apparent rate of Cl depletion, yet after 14 days all the animals were quite normal and the subsequent analyses of serum Cl showed a relatively small decrease in concentration.
(b) The active uptake of Cl
Cl absorbing mechanisms are, as might be expected, well developed in both ammocoete and adult. After washing out in distilled water larger animals of 4–5 g. have reduced the Cl concentration of 100 ml. of tap water (0·3 mM. Cl/1.) to about 0·06 mM. after 1 day, and one adult, which during 8 days in distilled water had lost 10μM. Cl, absorbed this ion from tap water diluted with 5 vol. of distilled water. Some evidence was also obtained of slight absorption from tap water diluted with 10 vol. of distilled water (approx. 0·03 mM. Cl/l.), but at these dilutions Cl analysis becomes uncertain.
For six adults, rates of absorption for the first day in tap water varied from 5 to 30 μM. and for fifteen large ammocoetes from 15 to 30 μM. The corresponding rates per g. were for the adults 5μM. and for the ammocoete 9μM., figures which are strikingly similar to those found by Krogh (1939) in Salmo irideus and Leuciscus rutilus, i.e. 7 and 9 μ M./g./day. For fluviatilisWikgren (1953) reports the rate of relative absorption as about 21 μ M./g./day, which is much higher than the maximum rate observed in planeri.
After 7–14 days in distilled water, absorption in tap water usually continued intermittently for several days, punctuated by periods of pronounced Cl losses. For eight ammocoetes of 3–6 g. the total net uptake of Cl during 14 days in tap water, following a similar period in distilled water, varied from 48 to 112μM. with a mean of 71 μ M. During the same period net losses to the tap water varied from 12 to 59/IM. with a mean of 36μM.
There does not appear to be any close quantitative relationship between total Cl uptake and the extent of the previous Cl depletion during the period in distilled water. Indeed, absorption was usually considerably in excess of preceding Cl losses. For the adult, the ratio of Cl uptake to Cl losses varied in seven animals from 1· 5 to 213, and in eight ammocoetes from 1· 0 to 6· 5. Some confirmation of the effect on internal Cl levels of washing out in distilled water, followed by a period in tap water, was obtained by determinations made on the serum of ammocoetes, both at the end of the distilled water period and after 14 days in tap water. In this experiment twenty-two animals were used, six being used as controls on which Cl determinations were made at the outset. The difference between the control group and the distilled water group, although not large appears to be significant (Table 5). Values obtained for animals after uptake in tap water are significantly higher than those of the control group, although some increase in Cl levels was to be expected in starving animals, even where they have not been previously washed out in distilled water. This marked excess of Cl uptake over previous Cl losses might be explained by supposing that, in the early stages of starvation, osmotically active materials are metabolized and the total concentration maintained by an increase in the Cl fraction. If this were so, then the uptake of Cl after washing out in distilled water would have two aspects : the replacement of the Cl ions actually lost to the distilled water and in addition the delayed substitution of Cl for other metabolites.
IV. THE PERMEABILITY OF THE INTEGUMENT TO WATER
In both species the osmotic uptake in tap water has been measured by weighing animals with the urino-genital papilla ligated. Under these conditions planeri adults survive for at least 4 hr., and the maximum weight increase for such a period was 22%. For at least the first 2 hr. the increase in weight appeared to be linear and the rates of uptake quoted are for this period. Fluviatilis survived for more than 30 hr. with weight increases of up to 30%, and over the first 24 hr. no decrease in the rate of uptake could be detected. For this species rates cited are those measured over the first period of 3 hr.
For nine adult planeri weighing from 2· 3 to 4· 7 g. the percentage weight increase per hour varied from 3· 8 to 7· 6 at 12–13° C., with a mean value of 5· 6. At the same temperature six adult fiuviatilis (21–54 g.) in October and November showed weight increases from 1· 0 to 1· 4%/hr. with a mean value of 1· 25, while two further animals tested in April gave increases in weight of 1· 7 and 1· 9%. From these figures the mean rates of urine flow in fresh water may be expressed as 1· 3 ml./g./day for planeriand 0· 3 ml./g./day for fiuviatilis. The urine output in fiuviatilis was measured directly by Wikgren (1953) at various temperatures by keeping the animals with only the anterior region of the body immersed in water. Interpolating from Wikgren’s data a value of 0· 3 ml./g./day is obtained for a temperature of 13° C., which is identical with that found by weighing. Such agreement provides support for the view that the branchial epithelium plays the major role in water transport.
Thus the permeability of the integument to water would appear to be about twice as great in planeri as in fiuviatilis.
V. THE EFFECTS OF SALINE SOLUTIONS
Observations have been made on the osmotic loss of weight in sea water and various dilutions of sea water with tap water. Experience showed that, even in solutions which were well below the level of concentration of the body fluids, there was in the case of adults and ammocoetes of planeri an initial loss of weight over and above the normal rate of metabolic loss for animals kept continuously in tap water. This excessive loss of weight was, however, compensated when animals were returned from the saline solution to tap water. Comparison of the behaviour of ligated and normal animals in saline solutions suggests that these initial losses are due to a delay in the reduction of the urine flow from its high fresh-water rate.
Although both ammocoetes and adult planeri survive well in diluted sea water, Ringer or even pure NaCl solutions of less than about 120 mM., they rapidly succumb in higher concentrations. In this respect the adult appears to be less resistant than the ammocoete. In sea water diluted to about 270 mM. the adult survived for about 6 hr., but pure sea water was generally lethal in 2–3 hr. By contrast, fluviatilis in October and November survived direct transfer to pure sea water for at least 6 hr. and in some instances tolerated indefinitely 270 mM. sea water. In both species a loss of weight of 20–25 % °f initial body weight appeared to be critical from the point of view of survival. Recovery, is, however, usually possible if the animal is returned to fresh water and the weight lost is rapidly regained. Indeed, there is a marked tendency for such animals temporarily to overshoot their initial weight.
In experiments with planeri, weighings were carried out in pure sea water and sea water diluted to approximately 360, 270 and 180 mM. With the exception of the second of these solutions, the same range was tested on fluviatilis in October and November. In the case of fluviatilis, weighings were made at two-hourly intervals up to 6 hr. or longer and, especially in the higher concentrations, there was a marked reduction in the rate of loss after the first 2 hr. Observations on planeri were made at hourly intervals and, in those solutions in which the animal survives for several hours, the rate of loss remained approximately linear. In Fig. 2 the osmotic loss/hr. expressed as a percentage of the initial weight is, in the case of planeri, that recorded after the first hour, and in the case of fluviatilis that recorded after the first 2 hr. in the solution. The osmotic gradients have been taken as the differences between the mean osmolar serum concentration (Table 2) and the estimated total concentration of the sea water.
Values obtained for the ammocoete and adult planeri seem to show a wider scatter than could be accounted for, either by weighing errors or individual differences in osmolar concentration, and losses recorded in early April were distinctly lower than those obtained the previous year in late April and May. Ammocoete values, while tending to be higher than those for the adult, show such a wide range that the differences are of doubtful significance. Examination of the observations in Fig. 2 suggests several points of interest:
The curves produced do not pass through the origin. There is little doubt that this may be attributed to the initial delay in the adjustment of urine flow.
For planeri the osmotic losses are lower than would be expected from the observed rate of osmotic uptake in fresh water. This can hardly be accounted for by the effects of the water losses on the internal concentration, since the same conditions apply (although in the reverse sense) in the determinations of osmotic uptake. It would be explained if it is assumed that the integument is readily permeable to the inward diffusion of ions.
For fluviatilis osmotic losses tend to be rather greater than expected. As this is especially true for the first period of 2 hr. it is thought to be due to the loss of mucus.
As a consequence of (2) and (3), while the rate of uptake in fresh water is over four times as great in planeri as in fluviatilis, the osmotic losses are in general no more than twice those observed in the larger species. If the interpretations which have been advanced are valid, the relative rates of uptake of the two forms have more significance than the rates of osmotic loss in relation to the physiological effects of direct transfer from fresh to salt water.
The swallowing of sea water, which is such an important factor in the regulatory mechanisms of marine teleosts, does not appear possible in the adultplaneri because of the occlusion of the foregut which takes place at metamorphosis (Weissenberg, 1926; Keibel, 1927), but the possibility remains that it might occur in fluviatilis when transferred from fresh water to sea water, as in this form closure of the gut does not appear to be complete until the animals become sexually mature in early spring. However, in view of the fact that, during the limited period of the present observations on these animals, osmotic losses in sea water were greater rather than less than expected, such a possibility would seem to be excluded.
Attempts were made, during late autumn and winter, to acclimatize fluviatilis to gradually increasing concentrations of sea water, but these failed to produce any evidence of regulatory processes. Starting from an initial concentration of about 80 mM. sea water, vapour-pressure and Cl determinations were made on the serum of pairs of animals killed at intervals as the concentration of the water was increased. The animals showed no obvious signs of abnormal behaviour until the water concentration exceeded 250 mM. and none died until the end of January when the concentration had reached 300 mM. Yet neither the values for Cl concentration nor the values for osmolar concentration gave any hint of regulation, and the animals became completely isosmotic beyond external concentrations of 200 mM. The last survivor died in February when the water concentration had reached 340 mM., after 6 weeks in water of 200 mM. and upwards (Fig. 3). As it is likely that the ability of these animals to regulate the internal concentration is lost soon after they enter the estuaries in the autumn and early winter, these attempts were presumably made too late in the season and were too prolonged.
Similar tests have been carried out on ammocoetes and adults of planeri and, except for the greater tolerance shown by the ammocoete to a raised internal concentration, the general trend was similar in both. Up to about 140 mM. the internal concentration remained above that of the water, but beyond this the animals approached a condition in which they were isosmotic and the concentration of the body fluids followed passively the increases in concentration of the external medium. The adult planeri began to succumb once the water reached 140 mM. and none survived beyond 160 mM. A few ammocoetes, on the other hand, survived in water of 200 mM., although they were by this time in an abnormal state.
VI. DISCUSSION
Apart from the total concentration of the blood, which is lower in planeri than in the anadromous lampreys, it appears that the differences in fat, water and Cl content between the ammocoete and adultplaneri and the adult fluviatilis are to be attributed to the period of starvation which, in both species, precedes sexual maturity. In both ammocoete and adult the effect of fasting is to increase the water content of the body and raise the Cl levels of the body fluids and tissues. The wide variations in the total Cl content of the ammocoete throughout the year and the changes in the Cl fraction of the total concentration shown by both planeri and fluviatilis emphasize the importance of the part played by Cl metabolism in the osmotic regulation of these animals in fresh water.
The increasing replacement of organic by inorganic constituents in the body fluids which accompanies sexual maturity is made possible by the ability to absorb ions from extremely low dilutions. Since the Cl ion is taken up in face of an increasing concentration of this ion in the body, it would not appear that the absorptive mechanisms are activated specifically by Cl depletion; a point which is further emphasized by the marked excess of Cl uptake over previous Cl losses which has been observed in animals transferred to tap water after a period of depletion in distilled water. The various observations which have been made on Cl metabolism are consistent with the view that it is changes in total concentration rather than Cl (or Na) ions that activates the absorptive mechanisms.
In planeri at least, the permeability of the skin and branchial surfaces to water is considerably greater than that recorded for teleosts in fresh water, even when allowances are made for differences in size and concentration gradients. For Salmo irideus and Carassius auratus the figures given by Krogh (1939) range between 0· 04 and 0· 1 ml./g./day, for Cyprinus and Anguilla (Smith, 1929) 0· 06–0· 15 ml., and for Ameirus (Marshall, 1934) 0· 3 ml. Moreover, this high rate of filtration in the kidney of the lamprey is achieved, not by a large number of discrete glomeruli, but by a small number of giant rope-like structures, formed by the fusion of separate glomeruli in the posterior part of the ammocoete kidney (Wheeler, 1899). The formation of large volumes of urine by the comparatively small filtration surface of such a kidney suggests a high hydrostatic pressure in the glomerular capillaries and lends added significance to the presence of valves at the origin of the segmental arteries (Young, 1950). Although no direct determinations have been made, the information obtained on urine flow and Cl losses leaves little doubt that the concentration of Cl in the urine must be very low, and that the kidney tubule is able almost completely to reabsorb Cl ions. Thus, taking a daily output of 3μM. Cl/g. with a urine flow of 1· 3 ml./g. and assuming all the Cl to be lost in the urine, the concentration of this ion could not be more than 2· 3 mM., which is less than that observed by Krogh (1939) in the urine of Salmo irideus and Carassius auratus.
Whatever may be the explanation for the reduced rate of osmotic uptake and loss of water through the integument of fluviatilis, there can be no doubt of its physiological significance for an animal which, in the course of its life history, changes from one medium to another. Fontaine (1954) has drawn attention to the relation between size and the amplitude of migration in those groups such as the Petromyzontidae and Salmonidae, which include both migratory and non-migratory forms, and indeed it may well be that increased size is in itself an important factor in the ability of the animal to cope with changes in salinity, through its effects on the relative surface area of the integument.
ACKNOWLEDGEMENTS
I should like to take the opportunity of thanking Prof. J. Z. Young, F.R.S., for his help and guidance on many occasions during the preparation of this paper and also Dr J. D. Robertson, who was good enough to read and criticize the manuscript. I am also indebted to many of my colleagues for their help in the course of my work and to others who have assisted me in the collection of the animals, particularly my wife and Mr J. R. O’Connor. Acknowledgements are also due to Dr J. D. Jefferson for his helpful suggestions in connexion with permeability and to the Governors of the Bristol College of Technology for the provision of facilities for this research.
REFERENCES
ADDENDUM
Since the preparation of this paper some information has been published by Morris (1956) on the osmo-regulatory ability of fresh-run and mature river lampreys subjected to gradually increasing concentrations of artificial sea water. These experiments confirm my own experience that there are marked differences in the water permeability of the integument and in tolerance to sea water of fresh-run and mature animals. In some fresh-run animals Morris found definite evidence of Cl regulation in diluted sea water (less than 270111M.) although the osmotic concentration of the plasma reached levels above those of the water. Urine flow was measured directly by Morris in two ways ; by cannulation after anaesthetiza-tion with chlorbutol and by a divided chamber method similar to that employed by Wikgren (1953). Using the latter method the rate of urine flow in fresh water was found to be 341· 9 ml./Kg./day at 16–18° C., a figure which agrees very closely with those obtained by both Wikgren and myself (when corrections are made for temperature). The cannulation method, however, gave a much lower figure (155-8 ml./Kg./day), which Morris believes to be the more reliable, but I find it difficult to accept his suggestion that the higher figures may be due in part to shock diuresis or to damage inflicted on the skin by the membranes used in divided chamber experiments. The fact that, in my own observations, the rate of weight increase in ligated animals has remained approximately linear for periods up to 30 hrs. and the close agreement reached by three separate observers using different handling techniques, seem to discount the influence of shock, while the effect of local skin damage would hardly be very serious, if as seems likely, the branchial epithelium is the main source of water uptake. Furthermore, in the absence of controlled experiments, it would not be safe to exclude the possibility of antidiuretic effects through the use of chlorbutol as an anaesthetic.